Viral infections are a principal cause of illness due to communicable diseases that affect the public at large. Of these, influenza viruses, including types A and B, are a significant factor responsible for causing respiratory symptoms as well as systemic malaise; other respiratory viruses include parainfluenza 1, 2, 3, and 4, respiratory syncytial virus, and adenovirus. The influenza viruses undergo rapid mutation of strains, producing pathogens with varying degrees of virulence and severity of symptoms. Recently, influenza infection has been as high as the fifth leading cause of death from acute respiratory disease in the United States (Morbidity and Mortality Weekly Report, 36 (1987) 2).
It is important to have available ways of diagnosing the presence of an influenza infection, and of distinguishing it from related viral and bacterial infections. Particularly among infants, the elderly, and those having compromised or deficient immune responses, early diagnosis of influenza can lead to appropriate treatment to minimize the risk of morbidity and mortality. Diagnosis of viral infection, such as infection by influenza virus, may be carried out by detecting the presence of unique moieties characteristic of the virus. Virus particles typically carry distinctive antigenic components on the exterior of the virion which may be detected by specific ligand-antiligand interactions, in particular by the use of an antibody specific for a viral epitope. Such interactions rely on the law of mass action, and for this reason may have limited sensitivity. Moreover, for viruses that mutate frequently, antibody recognition can be lost, thereby reducing the test's sensitivity and selectivity. Many virus particles additionally carry specific enzymatic activities on the virion particle. Influenza virus is such a virus; it is endowed with a virus-specific neuraminidase activity as an integral part of the virion which is exposed to the environment. Utilization of the enzymatic activity in such cases offers the potential for increasing the sensitivity of a detection method.
N-acetylneuraminic acid (sialic acid, Neu5Ac), whose structure is shown below with the numbering system used herein,
is the terminal saccharide residue of many complex carbohydrate side chains of cell surface glycoproteins. In this structure, the C2 position is the anomeric carbon, which is characterized by being part of a hemiketal group or the 2-ketoside moiety. The glycosidic linkage bonding Neu5Ac with the penultimate saccharide is the substrate of the neuraminidase activity of the influenza virion. The neuraminidase hydrolyzes the linkage, thereby cleaving Neu5Ac from the penultimate saccharide. Consequently, suitable synthetic substrates may be derivatives of Neu5Ac in 2-ketoside linkage with a detectable moiety. The moiety then provides a product, when the substrate is acted upon by the viral neuraminidase activity, that signals the presence and amount of influenza virus particles in a sample. Since the viral enzyme cleaves the substrate catalytically, the sensitivity of detecting the presence of the enzyme is greatly enhanced. For this reason the overall sensitivity of detection may be comparable to, and may even be improved over, that provided by antibody binding assays.
One method for detecting the presence of a virus through the reaction of an enzyme with a chromogenic substrate for the enzyme is described in U.S. Pat. No. 5,252,458. Another assay for the direct measurement of influenza neuraminidase was developed by Yolken et al. (J. Infectious Diseases 142 (1980) 516–523). Yolken et al. used the 4-methylumbelliferyl-2-ketoside of Neu5Ac as a fluorescent substrate to measure neuraminidase activity in preparations containing small quantities of cultivated virus as well as in some nasal wash specimens from human volunteers infected with the influenza virus. Yolken et al. suggested that “successful development of influenza neuraminidase might thus provide for a practical means of influenza diagnosis that is sufficiently rapid to allow for the institution of appropriate preventive and therapeutic interventions.” According to Yolken et al., calorimetric assays were insufficiently sensitive for clinical applications, suggesting instead that fluorimetric assays for influenza neuraminidase might be suitable for detecting the virus in clinical samples.
Pachucki et al. (J. Clinical Microbiology 26 (1988) 2664–2666) tested the 4-methylumbelliferyl-2-ketoside of Neu5Ac on clinical specimens collected from influenza patients. Due to its low sensitivity, the assay was not useful in detecting neuraminidase in clinical specimens. The assay did, however, identify 91% of virus-positive isolates 25 hours after inoculation of tissue cultures.
The use of modified Neu5Ac substrates can increase the specificity of the neuraminidase assay. In sialic acids, the C4 position has been reported to play an important role in enzyme-substrate interactions. Further, since it is known that salivary bacterial enzymes exhibit neuraminidase activity (Varki et al., J. Biol. Chem. 258 (1983) 12465–12471), it is essential to avoid these undesired enzymatic activities. It has, for example, been shown that ketosides of 4-methoxy-Neu5Ac are resistant towards certain bacterial sialidases (Beau et al., Eur. J. Biochem. 106 (1980) 531–540).
U.S. Pat. No. 5,252,458 to Liav et al. provides a direct chromogenic assay for detecting a virus, including influenza viruses and parainfluenza viruses, that include in the virion a characteristic enzymatic activity, such as neuraminidase activity. The method, which is implemented in a clinic or physician's office, includes incubating a clinical sample suspected of containing the virus with a solution of a chromogenic substrate. The samples typically are obtained by swabbing the pharyngeal, or nasopharyngeal surfaces. If the virus is present, a chromogen is cleaved from the substrate and then reacts with a precipitating agent. The resulting colored precipitate is concentrated for detection (e.g., concentrated as a colored spot on an appropriate substratum). The patent also discloses a kit for use in the clinic or physician's office that includes a filtration device for concentrating the colored precipitate into a spot. This patent provides synthetic routes for the synthesis of a precursor for chromogenic substrates that are useful in the diagnostic assay of viruses. Specifically the patent discloses syntheses for 4-alkoxy-N-acetylneuraminic acid.
U.S. Pat. No. 5,663,055 to Turner et al. discloses 4-modified Neu5Ac chromogenic substrates of viral neuraminidases for use in assays carried out in a clinic or a physician's office. The modification at position 4 includes hydrogen, fluorine, methoxy or ethoxy, and the substrate is sensitive to neuraminidases from influenza and parainfluenza viruses. Clinical samples are used in the assays, and distinct colors are produced as a result. The samples are typically pharyngeal, nasopharyngeal, or respiratory secretions and are collected from patients as, for example, wash, swab, or expectorate specimens.
U.S. Pat. No. 5,719,020 to Liav et al. discloses 4,7-di-O-alkylated chromogenic ketosides of N-acetylneuraminic acid for use as the substrate in assays that are specific for influenza A and influenza B. The assays are also generally performed in a clinic or physician's office. The chromophoric product may be precipitated and the colored precipitate concentrated for detection. Assays are also disclosed that permit discriminating between influenza A and influenza B on the one hand, and other viruses. U.S. Pat. No. 4,331,760 discloses chromogenic sulphonphthalein reagents impregnated in absorbent carriers for the detection of leukocytes in a clinical sample such as urine. The reagents are susceptible to action of esterases present in the leukocytes.
U.S. Pat. No. 4,526,753 discloses a card or strip containing a series of reagent impregnated matrices in sequence, separated by blank spaces, arrayed on a substratum. The strip may be exposed to a sample in order to cause reaction with reagents in the matrices, which may be the same or different.
U.S. Pat. No. 5,552,288 discloses an enzyme linked immunosorbent assay (ELISA) performed on a moist semi-solid sheet comprising chromogen agar paper impregnated with a substrate for an enzyme. Typically the enzyme is the enzyme conjugated to a detecting antibody used in the ELISA. The assay is intended for the detection of any of a variety of serum antibodies to antigens present on pathogens that may infect humans and animals. The assays can be carried out in settings lacking instruments, running water, or other conventional laboratory equipment. In the assay, a sample that may contain the antibody are bound to an immobilized antigen. An enzyme-labeled conjugate is added to bind the antibody. Then a paper coated with a solidified agar containing a substrate for the enzyme is added, and color from the substrate may develop if the assay is positive.
U.S. Pat. No. 5,160,436 discloses a multilayer test device including a permeable support membrane having a macroporous membrane on one side and a microporous membrane on its other side. Upon applying a fluid test sample to the macroporous membrane, free flow of fluid through this membrane and support membrane to the microporous membrane proceeds. This permits separation of incompatible reagents.
Although modification of the 4-position of Neu5Ac provides specificity between certain viral and certain bacterial neuraminidase activities, it is still desirable to obtain substrates which provide enhanced specificity between the various viral neuraminidase reactivities while maintaining the specificity between viral and bacterial neuraminidase activities. Such substrates allow, for example, high specificity for particular types of neuraminidase-containing viruses and allow better and more directed treatment regimes. Use of specific substrates thus allows for more accurate surveillance of viral infections and more focused medical intervention as appropriate. U.S. Pat. No. 5,719,020 provides chromogenic and fluorogenic 4,7-disubstituted N-acetylneuraminic acid substrates for viral neuraminidase activities. These derivatives provide further specificity or differentiation between the various viral neuraminidase activities while maintaining the specificity between viral and bacterial neuraminidase activities. In particular, the 4,7-modified Neu5Ac chromogenic substrates of U.S. Pat. No. 5,719,020 can distinguish between influenza type A and B viruses, on the one hand, and neuraminidases from other viral and bacterial pathogens, on the other. A chromogenic derivative of 4-O-methyl Neu5Ac, however, develops color when exposed to parainfluenza virus types 1 and 2, and mumps, in addition to influenza types A and B.
The synthetic procedures leading to the 4,7-disubstituted chromogenic Neu5Ac ketoside derivatives disclosed in U.S. Pat. No. 5,719,020 have certain difficulties associated with them. These include the provision of synthetic routes involving a large number of distinct chemical reactions. In addition, the 4,7-disubstituted derivatives are the minor component in a mixture of products, requiring extensive purification. These and other factors lead to considerable difficulties and disadvantages related to the synthesis of the 4,7-disubstituted chromogenic Neu5Ac ketoside derivatives. U.S. Pat. No. 5,719,020 addresses these difficulties and provides optimized synthetic procedures providing high yields of the desired pure 4,7-disubstituted chromogenic Neu5Ac ketoside derivatives.
Recently there has been intense interest in biological assay methods employing chemiluminescence using enzymatic triggerable 1,2-dioxetane molecules. Researchers have clearly established 1,2-dioxetanes as chemiluminescence compounds which can be used as reporters in ultrasensitive assays that can be conducted quickly, without resort to exotic conditions or elaborate apparatus, for the detection of a variety of biological materials. Both Tropix, Inc. (New Bedford, Mass.) and Lumigen, Inc. (Detroit, Mich.) have been especially active with regard to chemiluminescence using enzymatic triggerable 1,2-dioxetane. A number of such systems are described in, for example, U.S. Pat. Nos. 4,931,223; 4,931,569; 4,952,707; 4,956,477; 4,978,614; 5,032,381; 5,145,772; 5,220,005; 5,225,584; 5,326,882; 5,330,900; and 5,336,596; 5,707,559; 5,753,436; 5,756,770; 5,763,681; 5,773,628; 5,777,133; 5,783,381; 5,800,999; 5,827,650; 5,831,102; 5,840,919; 5,843,681; 5,847,161; 5,849,495; 5,851,771; 5,856,522; 5,866,389; 5,869,699; 5,869,705; 5,871,938; and 5,891,626, each of which are hereby incorporated by reference. Together, this wealth of patent literature addresses 1,2-dioxetanes stabilized by a typically polycyclic group, preferably spiroadamantane bonded to one of the carbons of the dioxetane ring, and a moiety bonded to the remaining carbon of the dioxetane ring which is electron sensitive, such that deprotection of the electron sensitive moiety, typically an aryl group, more preferably a phenyl or naphthyl group, leads to an anion, generally an oxyanion, which is unstable, and decomposes. Through decomposition, the O—O bond is broken, and a photon is generated. The same carbon atom to which this electron sensitive moiety is bonded may bear an alkoxy or other electron-active group. Methoxy is a preferred moiety. Assays using chemiluminescence derived from enzymatic triggerable 1,2-dioxetane are generally rapid, very sensitive, and convenient. The amount of light generated, or degree of chemiluminescence, can be correlated with the amount of the specific enzyme present. Assays using photographic film to detect and quantify the light generated are possible using such systems.
Nonetheless, methods utilizing the high sensitivity and selectivity of such chemiluminescence systems, including the 1,2-dioxetane chemiluminescence system, have not been used in detecting influenza and related viruses. Thus, it would be desirable to provide a viral analysis, and especially an influenza analysis, using chemiluminescence systems and especially the 1,2-dioxetane system. Moreover, because of the high prevalence of influenza in the population, it would be advantageous to have available an assay that can be carried out conveniently and, if necessary or desired, by a layperson in a nonmedical setting. The assays available at present generally fail to provide such an assay. Generally these assays are sufficiently complex or require such laboratory equipment that they must be conducted in a clinic or a physician's office. There is a need, therefore, to provide a test system, and a diagnostic test kit that is sufficiently simple in operation and other requirements that nonmedical personnel or layperson are able to conduct, for example, an influenza test in a nonmedical setting such as, for example, the home. Such a kit or method would ideally allow such a layperson to directly collect and test the sample from himself or from another individual such as a child, a person in poor health, or an elderly person in an nonmedical setting (e.g., the home, nursing home, and the like). The present invention satisfies these needs and objectives. The present invention can also be used in traditional clinical or medical environments by, if desired, by personnel with limited laboratory training.